Cell-based assay or bioassay development can range from cytotoxic assays, including apoptosis, to cell proliferation and metabolic assays. Cell-based assay development can also include high throughput screening assays and other custom bioassays used to characterize drug stability for GLP and GMP lot release, drug potency, and for drug purification and production support. Mechanisms of action, such as receptor binding, receptor activation, cell signaling, drug internalization and subcellular localization can be delineated in cell-based assays following treatment with drug compounds of interest. Bioassay development can encompass testing of conditioned medium, cell lysates or whole cells in a variety of formats including ELISA and immunohistochemical methods.
Exendin-4 (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO:1)) is a peptide found in the saliva of the Gila monster, Heloderma suspectum; and exendin-3 (HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO:2)) is a peptide found in the saliva of the beaded lizard, Heloderma horridum. Exendins have some amino acid sequence similarity to some members of the glucagon-like peptide (GLP) family. For example, exendin-4 has about 53% sequence identity with glucagon-like peptide-1(GLP-1)(7-37) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:22)). However, exendin-4 is transcribed from a distinct gene, not the Gila monster homolog of the mammalian proglucagon gene from which GLP-1 is expressed. Additionally, exendin-4 is not an analog of GLP-1(7-37) because the structure of synthetic exendin-4 peptide was not created by sequential modification of the structure of GLP-1. Nielsen et al, Current Opinion in Investigational Drugs, 4(4):401-405 (2003).
Synthetic exendin-4, also known as exenatide, is commercially available as BYETTA® (Amylin Pharmaceuticals, Inc. and Eli Lilly and Company). BYETTA® contains exenatide, a preservative (e.g., metacresol), a tonicity-adjusting agent (e.g., mannitol), and a buffer (e.g., an acetate buffer). A once weekly formulation of exenatide is currently in development and is described in WO 2005/102293, the disclosure of which is incorporated by reference herein. This once weekly formulation comprises exenatide and biodegradable polymeric (e.g., poly(lactide-co-glycolide)) microspheres, and is referred to herein as EQW (Amylin Pharmaceuticals, Inc., Eli Lilly and Company, Alkermes, Inc.).
There is a need in the art for new and improved bioassays for determining the potency and activity of pharmaceutical compositions containing GLP-1 receptor agonist compounds, such as BYETTA®, and for determining the potency and activity of GLP-1 receptor agonist compounds, such as exenatide. Cell-based bioassays that meet these needs are described herein.